Chlorosis

All wheat accessions were evaluated for seedling resistance to races QFCSC, QTHJC, RCRSC, RKQQC, TPMKC, TTTTF and TTKSK in a greenhouse and for adult plant resistance to bulked U.S. races (QFCSC, QTHJC, RCRSC, RKQQC and TPMKC) in the field at the USDA Cereal Disease Laboratory in St. Paul, MN in 2008. Field disease ratings were based on the percentage infection of the stems using the modified Cobb scale [28] when susceptible controls reached 60–70% severity. Seedling IT was scored using the Stakman scale [24] . Details on plant culture, inoculation methods, and scoring methods for the greenhouse and field experiments were described [29] .
To meet the data format required for association analysis, original seedling IT data were converted to a 0–9 linear disease scale as we described in a preliminary report [30] . Simple infection types were converted as follows: 0, 1−, 1, 1+, 2−, 2, 2+, 3−, 3 and 3+ were coded as 0, 1, 2, 3, 4, 5, 6, 7, 8 and 9, respectively. For lines with heterogeneous reactions, only the most prevalent IT was used. The semicolon symbol for hypersensitive fleck “;” was converted to 0. IT 4 was converted to 9. Special annotation code “S” for susceptible was converted to 9 and “S LIF” for low infection frequency was converted to 8. Special annotation codes “C” for extra chlorosis and “N” for extra necrosis were ignored. Double minus and double plus annotations were converted to single minus and single plus, respectively. Complex ranges such as ;12+ were first collapsed to ;2+. Then the first and last ITs of the range were converted and averaged with the first IT being double-weighted because the most prevalent IT is always listed first. Mesothetic reaction types X−, X, and X+ were converted to linearized scores of 4, 5, and 6, respectively. Y and Z mesothetic infection types were treated similarly to X. The conversion algorithm is implemented with examples as an editable Excel spreadsheet in Table S3. Each IT score was based on one replication comprising five to six seedlings per isolate, except for TTKSK, in which two replications were used for each accession and a mean value was used for association analysis.

For fungus inoculation, seedlings of G. hirsutum cv. YZ-1 that underwent VIGS treatment, and 3-week-old WT or transgenic tobacco plants were each gently uprooted, the roots were first rinsed in water and subsequently dipped into the spore suspension of 1×10⁵ conidia·ml⁻¹ for 1min. The plants were then replanted in fresh soil to monitor disease development. The fungal recovery assay in cotton was performed according to the method of Fradin et al. (2009) (link). Stem sections immediately above cotyledons were taken from cotton seedlings 10 days after V. dahliae inoculation and surface sterilized. The stem sections were subsequently cut into 5–8mm slices and incubated at 25 °C on potato dextrose agar. Cotton seedlings infiltrated with binary vectors pTRV-RNA1 and pTRV-RNA2 were used as the vector control, and the plants of WT and transgenic tobacco inoculated with water were used as mock controls. The disease index (DI) of Verticillium wilt was calculated according to the following formula:
Cotton leaves were classified in one of five levels of severity of disease symptoms during fungal invasion, and n denotes the disease level from 0 to 4 (Xu et al., 2012a). The resistance of tobacco to V. dahliae was evaluated by determining the extent of stunting (leaf size, weight of the plant) as well as the degree of damage to infected leaves (leaf chlorosis/wilting). The same formula above was adopted for disease index calculations in tobacco.
B. cinerea was incubated on potato dextrose agar medium for 4 days and then put onto leaves in concentric circles using a punch. A fungus with identical virulence was inoculated for 72h onto the second leaf in vitro, collected from the top of cotton seedlings. Growth of B. cinerea was estimated by the size of the necrotic lesions on the leaves.

During the fall of 2017, a total of 2,415 plant samples (S1 Table) were collected from grapevine populations with a historically high incidence of GLRaV-3 or with observable GLD symptoms such as interveinal reddening and downward rolling of leaf margins in red wine cultivars, and interveinal chlorosis and downward rolling of leaf margins in white cultivars [1 ]. These grapevine populations included: 1) the USDA National Clonal Germplasm Repository (NCGR) in Winters, CA, in which a previous study [18 (link)] identified plants infected with GLRaV-3 and originating from 12 different countries (1,206 samples); 2) the Davis Virus Collection (DVC) at University of California-Davis [21 ], which primarily consists of domestic GLRaV-3 isolates and is also the source of an isolate which is 99% identical to the GH24 isolate (109 samples); 3) the FPS pipeline of foreign and domestic introductions (417 samples); and 4) 89 vineyards in the main grape-growing areas of California including 77 samples from Napa Valley, 14 samples from Sonoma, 44 samples from San Luis Obispo, 39 samples from Monterey, 156 samples from Central Coast, 10 samples from Coachella Valley, 70 samples from the North Coast, 164 samples from the San Joaquin Valley and 109 samples from the Central Sierra region. Additionally, 45 samples from different GLRaV-3 collections outside of the USA were included in this study. These included 23 South African grapevines from eight different selections that represent the different GLRaV-3 variant groups present in that country; six samples originated from New Zealand, where several new GLRaV-3 isolates have been reported recently [8 (link)]; seven GLRaV-3 positive plants originated from Australia that showed mild leaf roll symptoms [22 ]; an asymptomatic GLRaV-3 infected ‘Pinot noir’ vine originating from Spain; and lastly, eight grapevine samples determined positive for GLRaV-3 by end-point RT-PCR and HTS during a study validating this technology for virus detection in Canada, including a sample (ON936) from a ‘Vidal Blanc’ vine infected with a putative new GLRaV-3 variant based on preliminary sequence analysis. Positive controls (described above) were included during the testing process, as well as a grapevine that tested negative by HTS for viruses and virus-like pathogens.
All the above-mentioned samples (leaf petioles or bark scrapings) were subjected to TNA extraction: 0.2 g plant tissue was homogenized in 2 ml of guanidine isothiocyanate lysis buffer (4 M guanidine isothiocyanate; 0.2 M sodium acetate, pH 5.0; 2 mM EDTA; 2.5% (w/v) PVP-40) and TNA extracts were prepared using a MagMAX-96 viral RNA isolation kit (Ambion, Austin, TX, USA) as per the manufacturer’s protocol. Subsequently, the integrity of RNA was verified using an 18S rRNA assay [18 (link)].

Leaf injury, manifested as chlorosis/necrosis, was assessed 15 d after spray application on approximately 150 leaves from each plot located from the middle portion of current-season shoots on both sides of rows on the outside of the tree canopy at a height of 1.5–2.0 m above the ground. Leaf burn was assessed visually on a scale of 1 to 5, where 1 represents lack of leaf blade injury and 5 indicates chlorosis/necrosis > 75% of the leaf area.
Cold injuries of buds and shoots were assessed on 50 current-season shoots per plot, located identical to those for the determination of leaf spray burn. The shoots were excised at the stage of dormancy/beginning of leaf bud swelling (BBCH 00/01, on 4 March 2021 and 2022) and placed into 20-L containers in which the level of tap water allowed the shoots to be immersed to a height of about 5 cm. The shoots were kept for 14 d at room temperature. Then, buds were dissected vertically to evaluate their section colour. Brown discolouration or necrosis was scored as cold damage, while the green colour was classified as alive. Cold bud damage was expressed as the percentage of browned/necrotic buds.
Cold injury of shoots was determined according to the electrolyte leakage (EL) method described by Wójcik [27 ]. Briefly, six shoots randomly selected from each container (replication) were rinsed with deionised water, dried with a paper towel and cut into approximately 2 cm segments without buds. Then, 60 g of cut shoots were placed into a 200 mL glass beaker containing 40 mL of deionised water and incubated in a water bath (LWTc; WSL Świętochłowice, Poland) at room temperature for 24 h. The initial electrical conductivity (EC1) of the medium was measured using an electrical conductivity analyser (Orion Star A2/2; Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the samples were heated to 100°C for 30 min to allow maximum leakage from shoot segments. After cooling to room temperature, the electrical conductivity was remeasured (EC2). The electrolyte leakage was calculated using the equation EL (%) = EC1/EC2 ×100.

The wheat cultivar Henong 6425 was chosen as the experimental material and was acquired from Tianjin Academy of Agricultural Sciences. Henong 6425 is susceptible to powdery mildew. The wheat seedlings were grown in hydroponics as described previously [38 (link)]. At the two-leaf stage, uniformly growing seedlings were chosen for subsequent PW inoculation. The fresh conidia of Bgt, whose virulence type is E09, on heavily diseased seedlings were shaken off onto the second leaves, which were flattened. Leaves were at 0, 3, 6, 12, 24, and 48 h after PW infection, immediately frozen in liquid nitrogen and then stored at -80 °C for subsequent RNA extraction.
A cDNA fragment of 131 bp (+ 1612 bp to + 1742 bp) was employed to acquire the TaCDPK27-silenced vector. TaCDPK27-silenced wheat seedlings were acquired with barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS), as described by Yue et al. (2022) [14 (link)]. Control (containing wild-type (CK) seedlings and fourth leaf-stage BSMV-VIGS-inoculated seedlings that displayed chlorosis (the materials of which included BSMV-VIGS-GFP-inoculated seedlings (γG) and TaCDPK27-silenced seedlings) were inoculated with PW causal agent conidia. Fungal structures were subsequently stained withCoomassie brilliant blue and visualized via microscopy.

P. medicaginis is a homothallic species. Ten isolates of P. medicaginis were used (as a mixture) in all experiments, storage, and isolate culturing as described in Bithell et al. (2022) (link). Prior to inoculum production, each isolate was passaged through plants in a glasshouse using the very susceptible chickpea variety Sonali to ensure pathogenicity. With the use of low-strength V8 media (100 ml of V8 juice, 10 g of agar, 2.5 g of calcium carbonate, and 900 ml of Milli-Q water), an oospore suspension was prepared by macerating cultures with a hand-held Braun 600W blender and then added to flooded (Milli-Q water) cups of seedlings in potting mix, which were then drained after 48 h. After the observation of wilting, chlorosis, and canker development on the seedlings, stem tissue at the margin of the canker was used to re-isolate the pathogen on corn meal agar. Cultures were hyphal tipped and then grown on low-strength V8 media. Subcultures of these freshly passaged isolates were used to produce 90-mm-diameter Petri dish cultures of each isolate, which were grown in the dark at 21°C–23°C for at least 6 weeks prior to mixing with Milli-Q water (10% V/V) and macerating using a hand-held Braun 600W blender for approximately 3 min. Average oospore concentrations for each isolate were determined using counts under a 20 * 50-mm coverslip to prepare inoculum mixtures containing equal oospore concentrations.